The definitive characteristic that distinguishes the heart tissue environment from other organs is its rhythmic physical contraction. A comprehensive understanding of how cardiac myocytes maturate in this unique biophysical environment at fetal stages is critical for both deeper understanding of the pathogenesis of congenital heart disease and application of PSCs to cardiac regeneration. This proposal will take an interdisciplinary approach combining stem cell biology, quantitative physical method, and bioinformatics to apply the findings from mouse developmental biology to the engineered maturation of human PSC-derived cardiomyocytes and zebrafish. We have established a foundation for multifaceted solutions to various technical hurdles over the past year: selection of the optimal human embryonic stem cell (hESC) lines, optimization of the readout of the functional/genetic/epigenetic maturation of hESC-derived cardiomyocytes using cellular force spectroscopy (CFS) and mechanical imaging interferometry (MII) unique to our labs, structural characterization via super-resolution confocal microscopy (STED), and application of optimized electro-mechanical forces using multielectrode arrays (MEA). Our data suggest that both stiffer microenvironment and applied electrical stimulation enhance cardiac differentiation of hESC lines. Thus, the cardiomyocytes maturate to generate unique biophysical activities, which in turn serve to the maturation of the migrating cardiac progenitors, forming a positive feedback loop. To test the concept that electro-mechanical cues and genetic programs mutually reinforce each other to induce the progressive maturation of cardiac progenitors and examine how biophysical cues are translated into genetic/epigenetic mechanisms and vice versa, we will apply quantitative physical methods in vitro to hESC-derived cardiomyocytes. Towards the in vivo application of this concept, we also propose to establish zebrafish experimental system. Successful completion of this proposal will elucidate a fundamental principle of cardiac maturation and unlock the maturation block of PSC-derived cardiomyocytes, which is a critical barrier between current cardiac stem cell research and future regenerative medicine.